The long-term goal of this project is to define the role of the novel lipid pathway mediated by sphingosine kinase (SK) and its bioactive product sphingosine-1-phosphate (S1P) in cancer, and to establish components of this pathway as potential novel targets for cancer therapy. Studies from the previous funding period have led us into a novel exciting direction on the role and regulation of SK/S1P in cancer. S1P is generated by breakdown of the sphingolipid ceramide to sphingosine, followed by phosphorylation by SK, and it then mediates/modulates several important biologic activities, including mrtogenesis, cell survival, and angiogenesis. During the previous funding period, we established that SK1 is significantly overexpressed in several cancers and is upregulated by hypoxia. Moreover, we discovered a novel mechanism of regulation of SK1 by proteolysis in response to genotoxic stress in a p53-dependent manner, and showed that loss of SK1 led to growth arrest and apoptosis of cancer cells. These data have led us to propose the following hypothesis: that activation of p53 causes the loss of SK1, that this SK1 proteolysis mediates p53 tumor suppressor function, and that the SK1/S1P pathway mediates null/mutant p53-induced cancer. To test this hypothesis we propose the following specific aims: 1} To establish that p53 regulates the SK1/S1P pathway and decipher the mechanisms involved by A) Demonstrating that SK1 is proteolyzed in response to the p53 activators, UV radiation and DMA damaging chemotherapeutic agents in cells. B) Determining if p53 is required for SK1 proteolysis in cells. C) Determining the expression of SK1 in p53 null and mutant tissues and compare to matched normal tissues from p53 null/mutant animal models and human cancer tissues. D) Determining the mechanism by which activation of p53 leads to SK1 proteolysis, and the sites of cleavage. E) Evaluating the consequences of induction of p53 and SK1 proteolysis on the cellular sphingolipid profile. 2) To determine if the persistence of the SK1/S1P pathway is necessary to mediate null/mutant p53 tumor progression pathways in cells and in vivo by A) determining the role of the SK1/S1P pathway in mediating p53 induced growth responses in cells. B) To develop through synthesis more potent and specific inhibitors of SK1. C) To evaluate these compounds as to their ability to induce apoptosis/senescence-like growth arrest in null/mutant p53 growth in cells. D) To determine the role of SK1/S1P in null/mutant p53 mediated cancer in vivo using SK1 K/O mice crossed with p53 null or mutant mice and determining if they are protected from tumor formation. These studies may provide a novel molecular link between the p53 and the sphingolipid pathways of cell stress regulation, both of which play key roles in regulating growth, apoptosis, and senescence. Lead compounds from this study may pave the way towards specific therapeutic development targeting SK1